The maintenance of cell volume is a fundamental property of all eukaryotic cells. The cell membrane of animal cells is highly permeable to water, the cell volume will be determined by the cellular content of the osmotically active solutes and by the osmolality of the extracellular fluid. The intracellular and extracellular environments are significantly different. A combination of active and passive transport processes are involved in cell volume regulation. Viable and healthy cells maintain constant cell volumes under resting conditions and are capable of counteracting volume perturbations by performing volume recovery processes.
Swollen cells tend to reduce their volume often by losing KCl and by concomitant loss of cell water. This is known as a "regulatory volume decrease (RVD). Similarly, shrunken cells show the capacity to increase their volume to initial or normal healthy values by taking up extracellular KCl and the concomitant uptake of cell water. This is known as "regulatory volume increase" (RVI).
The process of volume regulation involves a large number of transport systems and signaling mechanisms. Now, the only way to measure the "health" of cells is to wait for cell death and measure live versus dead cells by dye exclusion or the ability to grow into colonies. Similarly, the only way to measure apoptosis has been through a difficult and cumbersome process of cell sorting. The process of cell sorting does not lend itself to high throughput mechanisms. However, there is a need in the art to develop high throughput screening mechanisms to measure cell "health" and not just cell death and to measure the beginnings of the process of apoptosis rapidly and inexpensively. The present invention, using an epilepsy screening model, developed a novel and inexpensive high throughput screening system for cellular health and apoptosis that is applicable to evaluate compounds for a variety of treatment indications wherein the underlying diseases and their pathology (on a cellular level) is caused by poor cell health of certain cell types and apoptosis.
Two primary features that characterize epileptiform activity are hyperexcitability and hypersynchronization (Schwartzkroin, in The Treatment of Epilepsy: Principles and Practice, E. Wylie, Ed. Lea & Febiger, Philadelphia, 1993, pp. 83-98.). Although either of these features alone might be sufficient for the development and spread of epileptiform activity, it has not been possible to dissociate them in experimental models of epilepsy. Some studies have suggested that nonsynaptic mechanisms might be sufficient to produce hyperexcitability and/or hypersynchrony. For example, exposing hippocampal slices to calcium-free medium abolished synaptic transmission but produced synchronized burst discharges in a CA1 subfield (Jefferys and Haas, Nature 200:448, 1982; Taylor and Dudek, Science 218:810, 1982; Konnerth et al., Exp. Brain Res. 51:153, 1984; and Richardson et al., Brain Res. 294:255, 1984). Synchronized activity may be mediated by ephaptic interactions among the densely packed CA1 neurons (Dudek et al., Basic Mechanisms of the Epilepsy's: Molecular and Cellular Approaches, Delgado-Escueta et al. Eds. Raven Press, New York 44:593-617, 1986).
A major component of volume regulation of the ECS (extracellular space) under normal and pathological conditions is glial dependent (Kimelberg and Ransom, in Astrocytes: Cell Biology and Pathology of Astrocytes, Fedoeroff and Vernadakis, Eds. Academic, New York, 3:129-166, 1986). A furosemide-sensitive Na--K--2Cl cotransporter plays a major role in cell swelling and volume-regulation of astrocytes in cell culture (Geck et al., Biochim. Biophys. Acta 600:432, 1980; Walz et al., J. Cerebr. Blood Flow Metab. 4:301, 1984; and Kimelberg and Frangakis, Brain Res. 361:125, 1985). Changes in external K+ concentrations alter intracellular KCl concentrations in astrocytes. The changes are thought to act through furosemide-sensitive Na--K--2Cl cotransport, accompanied by osmotically-driven water movement. Furosemide blocks glial cell swelling (Kimelberg and Ransom, in Astrocytes: Cell Biology and Pathology of Astrocytes, Fedoeroff and Vernadakis, Eds. Academic, New York, 3:129-166, 1986; Geck et al., Biochim. Biophys. Acta 600:432, 1980; Walz and Hertz, J. Cerebr. Blood Flow Metab. 4:301, 1984; and Kimelberg and Frangakis, Brain Res. 361:125, 1985). However, the concentrations that furosemide acts are well beyond those than can be achieved in a clinical setting. Therefore, there is a need in the art to device a system, including hardware and software, that is able to screen for active drug compounds in culture in a high-throughput screening assay system. The present invention utilized furosemide to develop such a screening system.
Glutamate plays a vital role in the normal functioning of neurons. It is the main excitatory neurotransmitter in the central nervous system (CNS). The normal function of glutamate, as a means of communication from one neuron to the next, breaks down in certain disease states. Damage to the CNS associated with ischemia, most commonly in cerebrovascular embolitic disease or stroke, is a direct result of hypoxia or deprivation of metabolic intermediates. Glutamate is excessively effluxed during hypoxia by ischemic neurons, which in turn, activates pathways in post-synaptic neurons leading to acute cell swelling and later, cell death. Astrocytes maintain ionic, amino acid neurotransmitter and water homeostasis in the extracellular space of the brain. When applied to a culture of astrocytes, glutamate (at concentrations from 50 .mu.M to 1 mM) caused cell swelling (Juurlink et al., Can. J. Physiol. Pharm. 70:5344-9, 1992). There is a need in the art to discover drug products administered as early as possible after the ischemic event, that can attenuate the damaging action of glutamate and decrease morbidity associated with stroke and other neurodegenerative disorders.
Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Apoptosis plays an important role in embryogenesis and development and in tumor growth. Apoptosis is characterized by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytic cells to prevent an inflammatory reaction to degredative cell contents. Apoptosis has not been identified in vivo due to problems of heterogeneity and short half life of an apoptotic cell. Apoptosis has been characterized in vitro using dyes and flow cytometry (Dive et al., Biochem. Biophys. Acta 1133:275-85, 1992). However, flow cytometry is an extremely time consuming and labor-intensive process that cannot be used for high-throughput screening procedures that needs to investigate large numbers of candidate drug products. Therefore, there is a need in the art to find drug candidates from large libraries of compounds that appear to have therapeutic activity to reverse or prevent the apoptotic process and thus exhibit potential therapeutic utility in treating or preventing the progression of degenerative diseases such as Alzheimer's Disease (AD) and other neurodegenerative diseases.
AD is characterized by the accumulation of amyloid plaques, neurofibrillary degeneration, and accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the beta amyloid protein (BAP), which is a proteolytic fragment of the membrane-associated amyloid precursor protein of 39-43 amino acids in length. The neurotoxicity of BAP has been observed in vitro in cell culture and it appears to induce an apoptotic death. Similarly, glutamate will cause cell death of neuronal cells in culture and first cause cell swelling.
There are new technologies being developed that involve creating functioning cells that secrete biologically active materials. Such cellular cultures are encapsulated in polymeric materials that avoid problems of antigen recognition and rejection. However, in manufacturing such "living" therapeutic devices, it is important to check on the quality of such devices by measuring the health and viability of the cells encapsulated during various stages of the manufacturing process and prior to use. Therefore, there is a need in the art to develop processes and devices for measuring the health and viability of encapsulated cells of living cellular implants.